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Preparation of immunoadsorbent which interacts specifically with cortisol binding globulin cbg and its application for the interaction between cbg and cortisol evaluation of association constant ka between cbg and cortisol in normal subjects



Preparation of immunoadsorbent which interacts specifically with cortisol binding globulin cbg and its application for the interaction between cbg and cortisol evaluation of association constant ka between cbg and cortisol in normal subjects



Folia Endocrinologica Japonica 63(5): 695-701



We have developed a simple method for the evaluation of the association constant (Ka) between cortisol binding globulin (CBG) and cortisol using immunoadsorbent and a small amount of serum. Immunoadsorbent was prepared as follows; gamma-globulin fraction was obtained from goat anti-human CBG antiserum by salting out using 50% ammonium sulfate, followed by coupling to CNBr-activated Sepharose 4B. Immunoadsorbent thus prepared was used for the evaluation of Ka value between cortisol and CBG. Sera obtained from 5 healthy subjects (3 males and 2 females) were treated with charcoal (Norit A) to remove endogeneous cortisol. Each of 10 .mu.l of serum was incubated with a constant amount of 125I-cortisol and different concentrations of unlabelled cortisol for 12 hours at 25.degree. C, pH 7.4. The, 50 .mu.l of anti-CBG-Sepharose 4B (immunoadsorbent) was added to each incubation mixture, followed by incubation for another 4 hours at 25.degree. C. After centrifugation, radioactivity of the precipitate was counted and the results were analyzed using Scatchard plot. The Ka value between serum CBG and cortisol in 5 healthy subjects was 1.70 .times. 108 M-1 (SD = 0.24), which is comparable with that of a previously reported value using purified CBG from a large amount of serum. Interassay variation of this method was satisfactory with CV value of 10.1%. Our present method is considered to be clinically relevant, since it does not require purification of CBG from serum, and a relatively small amount (total amount, less than 200 .mu.l) of serum is enough to evaluate Ka value between CBG and cortisol.

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Preparation of an immunoadsorbent interacting specifically with cortisol-binding globulin (CBG) and its application to the interaction between CBG and cortisol--evaluation of the association constant (Ka) between CBG and cortisol in normal subjects. Nihon Naibunpi Gakkai Zasshi 63(5): 695-701, 1987

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